A novel TACSTD2 mutation identified in two Chinese brothers with gelatinous drop-like corneal dystrophy.

PURPOSE
To identify the molecular defect causing gelatinous drop-like corneal dystrophy (GDLD) in two Chinese brothers and report the morphological evaluation of GDLD by laser scanning confocal microscopy and Fourier-domain optical coherence tomography (OCT).


METHODS
Genetic analysis included polymerase chain reaction (PCR) amplification and direct nucleotide sequencing of the coding region of the tumor-associated calcium signal transducer 2 gene (TACSTD2) in DNA from the two brothers and their relatives. Laser scanning confocal microscopy and Fourier-domain OCT were performed on the left cornea of the younger brother.


RESULTS
We report a novel in-frame mutation of TACSTD2, c.526_576del 51, in the two brothers with GDLD. The identified molecular defect cosegregated with the disease and was not found in 50 unaffected individuals. The morphological evaluation on GDLD highlighted pathological observations at the level of epithelium and anterior stroma. The epithelial cells of GDLD cornea were irregular in shape and often elongated. Large accumulations of brightly reflective amyloid material was noted within or beneath the epithelium and within the anterior stroma.


CONCLUSIONS
The newly identified mutation expands the spectrum of mutations in TACSTD2 that may cause pathological corneal amyloidosis. Observations by in vivo confocal microscopy and Fourier-domain OCT were consistent with the histopathologic descriptions of GDLD.

Gelatinous drop-like corneal dystrophy (GDLD; OMIM 204870) is an extremely rare autosomal recessive disease characterized by the deposition of amyloid material in the subepithelial space of the cornea. It has an estimated prevalence of 1 in 300,000 in Japan. GDLD patients have also been observed in India, the United States, Europe, Tunisia, Turkey, and Vietnam [1]. Clinical manifestations of GDLD most often appear in the first decade of life with a bilateral, axial, elevated, mulberry-like gelatinous lesion. The amyloid depositions in the central cornea, which appear in the early stage of disease, will increase in number and depth and coalesce, leading to a progressive opacification of the cornea and resulting in significant decrease in vision, photophobia, lacrimation, and foreign body sensation. Eventually, penetrating or lamellar keratoplasty, photoablation, or keratectomy is required to recover corneal clarity and visual rehabilitation. Unfortunately, symptoms generally recur within a few years after intervention, and repeated keratoplasties are often performed [2].
Genetic linkage analysis of 20 GDLD families in Japan established a 2.6 centimorgan (cM) interval on chromosome 1p as the chromosomal segment harboring the putative causal gene [3]. Later, membrane component, chromosome 1, Correspondence to: Professor Liya Wang, Henan Key Laboratory of Keratopathy, Henan Eye Institute, No.7 Weiwu Road, Zhengzhou, 450003, PR China; Phone: +86-371-65580901; FAX: +86-371-65952907; email: wangliya55@tom.com surface marker 1 (M1S1), previously termed GA733-1 or TROP2, was identified as the causative gene located within this region [4]. M1S1, in agreement with the Human Genome Organization (HUGO) nomenclature, has been renamed TACSTD2 (tumor-associated calcium signal transducer 2) [5]. TACSTD2 consists of a single exon spanning about 1.8 kb of genomic DNA. It codes for a protein of 323 amino acids, which is a monomeric cell surface glycoprotein expressed in the cornea, multistratified epithelia, and trophoblasts and is found in most carcinomas with high protein expression levels [4,6]. The physiologic function of TACSTD2 has not been completely elucidated, however it is hypothesized that the protein can act as a calcium signal transducer [6].
Approximately 24 different TACSTD2 mutations have been reported to date, predominantly in patients from Asia [7,8]. Herein, we present the clinical, histopathologic, and genetic assessment of two Chinese brothers with GDLD in which a novel TACSTD2 mutation was identified. Our findings add to the allelic heterogeneity of this rare form of inherited corneal disease.

METHODS
This study was approved by the Institutional Review Board of the Henan Eye Institute (Zhengzhou, PR China). Informed consent in accordance with the Declaration of Helsinki was obtained from the patients and their family members who participated in this study.
GDLD case 1-A 39-year-old Chinese man (the proband) was referred to our department in August 2007 with bilateral GDLD. He had complaints of defective vision since childhood. His vision had worsened in the past 10 years. He had recurrent episodes of lacrimation and photophobia in both eyes. His visual acuity was limited to counting fingers at 1 m in both eyes at the time of presentation. Under slit-lamp examination, the right cornea showed diffuse opacities with multiple grayish-white nodular elevations located in the subepithelial area whereas his left eye showed band-shaped opacities in the interpalpebral area of the cornea with several gelatinous prominences above the band-shaped opacities ( Figure 1A,B). Details of the anterior chamber and lens were not visualized. In September 2007, we performed a lamellar keratoplasty on the right eye using a fresh donor cornea. At the most recent examination, the cornea showed no recurrence of GDLD with a best-corrected visual acuity (BCVA) of 20/40. GDLD case 2-A 49-year-old man, the elder brother of the proband, was seen in our department in October 2007 with bilateral GDLD. He presented with the same symptoms as the proband. Visual acuity was counting fingers in the right eye and hand movements in the left eye. The patient didn't seek medical attention until this point. Examination revealed yellowish corneal lesions with an irregular surface throughout the entire stroma and superficial vascularization in the right eye whereas his left eye showed the characteristic findings of GDLD such as multiple milky-white, opaque, semiglobate prominences ( Figure 1C,D).
These two brothers had no significant systemic diseases. Ophthalmic examinations of their mother and siblings didn't reveal any ocular abnormalities, and there were no known similar familial ocular conditions according to the report from other family members. The parents of the brothers were unaware of any consanguinity, but both parents came from the same small village. The village is isolated, and the population flow is very small.
Mutation analysis: Molecular genetic analyses were performed on the two brothers and the unaffected family members (members I:2, II:1, II:5, III:2, and III:5 from this family; Figure 2A). Fifty unrelated, healthy Chinese subjects were examined as controls. Genomic DNA was obtained from peripheral blood leukocytes according to standard procedures. For amplification of the single exon of TACSTD2, polymerase chain reaction (PCR) was performed, and primer pairs were used as designed by Akhtar et al. [9]. Their sequences are as follows:   Taq polymerase (Qiagen, Hilden, Germany). GC buffer was provided by Takara Biotechnology (Dalian) Co. Ltd (Liaoning, China) and designed for amplification of templates having complex secondary structure or high GC content. Amplification reactions were performed under the following conditions: 4 min of denaturation at 95 °C followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 50−55 °C for 30 s, extension at 72 °C for 30 s, and a further extension step at 72 °C for 8 min in a Gene-Amp PCR system 9700 (Perkin-Elmer Applied Biosystems, Foster City, CA). For direct sequencing, PCR products were purified using the QIAquick PCR purification kit (Qiagen), cycle sequenced with a Big Dye Terminator cycle sequencing kit (Perkin-Elmer Applied Biosystems), and directly sequenced on both strands using an automatic DNA sequencer (ABI Prism 377 Genetic Analyzer; Perkin-Elmer Applied Biosystems) according to the manufacturer's instructions.
Nucleotide sequences were compared with the published cDNA sequence of TACSTD2 (GenBank accession number NM_002353.2). The novel mutation found in the TACSTD2 coding region was confirmed by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis.
Histopathologic analysis: Corneal tissue, excised at the time of a lamellar keratoplasty in the younger brother, was available for histopathologic examination. The corneal button was sectioned and analyzed with light microscopy after staining with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and Congo red stains.
Confocal microscopy and Fourier-domain OCT analysis: In this study, we coupled in vivo confocal microscopy and Fourier-domain optical coherence tomography (OCT) to evaluate the morphological changes of the cornea in a patient with GDLD and compared the results with the clinical ocular surface findings and histopathologic examination.
After a detailed explanation, a laser scanning in vivo confocal microscopy with a diode laser with a wavelength of 670 nm (HRT3/Rostock Cornea Module; Heidelberg Engineering, Heidelberg, Germany) was performed on the left cornea of the younger brother. Two-dimensional confocal images of the different corneal layers were acquired. Subsequently, the left cornea of the younger brother was scanned three times with two scan patterns, the line scan and the cross-line scan, by a high-speed, high-resolution, Fourierdomain optical coherence tomography (Optovue Inc., Fremont, CA).We chose the best of the three scans for analysis.

RESULTS
Mutation analysis: Two sequence variations were identified in TACSTD2 of the brothers with GDLD. In the two brothers (case1-II:7 and case2-II:3), we found a 51 bp deletion between nucleotides 526 and 576 (526-576del 51) in coding region of TACSTD2 ( Figure 2B), which led to a loss of 17 amino acids at codons 176-192. This novel mutation was observed in the homozygous state in the affected brothers and in a heterozygous state in the proband's mother, daughter, and nephew (I:2, III:5, and III:2). This mutation was confirmed using the restriction enzyme, BstXI (Takara Biotechnology). PCR products (681 bp) harboring the homozygous mutation were digested with BstXI. Wild amplicons were digested into 468 bp and 213 bp DNA fragments while the mutant could not be cut as the mutation erased the enzyme recognition site. Therefore, digested amplicons from patients with a homozygous mutation had only one electrophoretic band (681 bp), but those from heterozygous unaffected family members yielded 681 bp, 468 bp, and 213 bp fragments ( Figure 2C).
Sequencing of the 382 bp PCR product of TACSTD2 in the affected brothers revealed a 12 bp deletion from nucleotide position 772-783 [772-783del(ATCTATTACCTG)], resulting in a loss of four amino acids at codons 258-261. Also, in the place of the missing sequence, an insertion of T (772insT) was found. This combined mutation was confirmed by analysis of both the forward and reverse sequence data and was heterozygous in the patients and the unaffected family members (I:2, III:5, and III:2; Figure 2D).
None of the above identified mutations were detected in the control population of 50 unaffected, unrelated, healthy volunteers as determined by restriction enzyme digest. Histopathologic analysis: Histopathologic analysis of the corneal button that was excised during a lamellar keratoplasty from the proband revealed areas of irregular epithelial hyperplasia, areas of epithelial atrophy, the destruction of Bowman's layer, and large deposits of eosinophilic material that had positive staining of PAS and Congo red. The corneal stroma was vascularized and showed a chronic inflammatory cell infiltrate ( Figure 3A-C). The deposits manifested different patterns depending on their position. Subepithelial deposits formed massive clumps that appeared to push up the epithelial layer to form mulberry-like patterns. Higher magnification of the area just beneath the epithelium revealed band-like sections with many spikes vertical to the basement line or amyloid materials. All the Congo red-positive deposits within or beneath the epithelium and within the anterior stroma manifested birefringence under polarized light ( Figure  3D,E).

Confocal microscopy and Fourier-domain OCT analysis:
Laser scanning in vivo confocal microscopy revealed that the epithelial cells of the GDLD cornea were irregular in shape and often elongated. This was in contrast to normal epithelial cells, which appeared polygonal in form and were 30-40 µm in diameter ( Figure 4A,B). Furthermore, a mild disorganization of the overall epithelial architecture was noted ( Figure 4C,D). The in vivo laser scanning confocal microscopic image at the level of the Bowman's membrane showed a very small number of sub-basal nerves with increased background intensity (Figure 4E,F). Large accumulations of brightly reflective amyloid materials were noted within or beneath the epithelium and within the anterior stroma. The nuclei of keratocytes were poorly identifiable because keratocytes were embedded in the extracellular matrix of increased reflectivity ( Figure 4G,H). No evident abnormalities could be detected in posterior cornea ( Figure  4I,J) .
The images acquired from Fourier-domain OCT, showing the reflectivity of all corneal layers, indicated that the main presence of amyloid deposits was localized within the anterior stroma, extending from the epithelium layer to a depth of 150-230 µm and that the highest density of amyloid deposits seemed to be mainly in the anterior stroma, which is within the first 100 µm of the corneal depth ( Figure 5).

DISCUSSION
The autosomal recessive form of gelatinous drop-like corneal dystrophy is a rare eye disorder caused by mutations in TACSTD2. Molecular genetic analysis of patients from diverse ethnic backgrounds showed both genetic and allelic heterogeneity for GDLD [10][11][12][13][14]. Herein, we report the molecular genetic analysis of TACSTD2 in two Chinese brothers with GDLD in which two TACSTD2 mutations were identified.
A 526-576del51 mutation and a 772-783del+772insT mutation were revealed by direct sequencing of the coding region of TACSTD2 in these two brothers. The 772-783del +772insT mutation was described in a Vietnamese patient who was homozygous for this mutation [13]. In contrast, our patients and their family members (I:2, III:5, and III:2) were all heterozygous for this mutation in TACSTD2. Our patients are from an isolated village in the central region of China and have no ethnic relationship with the Vietnamese. However, the 526-576del51 mutation was homozygous in the GDLDaffected brothers and heterozygous in their family members (I:2, III:5, and III:2), indicating that it was cosegregated with the phenotype. Such genetic alteration was excluded in the control population. Thus, this homozygous mutation was expected to cause GDLD in the two brothers. To date, only three patients with genetically mapped GDLD were reported from China having the homozygous or compound heterozygous mutation [7,14]. The previous Chinese GDLD patient with the Q118X/Y184C mutation lived in northern China whose ethnic relationship with the Japanese was unclear. Two other GDLD patients are from two unrelated consanguineous families in the southern and southeastern regions of China. The two different mutations identified in these two patients are both homozygous frame-shift mutations, and the sites of the mutations are unique. These consisted of two insertions/deletions of a nucleotide, causing a shift in the translational reading frame and introducing a premature termination codon not far downstream of the mutation site.
The 40 kDa TACSTD2 protein contains an epidermal growth factor (EGF)-like repeat, a thyroglobulin type 1A (TY) repeat, a transmembrane domain (TM), and a phosphatidylinositol (PIP2)-binding site, harboring phosphorylatable serine and threonine residues near the COOH-terminus. The single transmembrane segment extends from position 275-298 of the amino acid sequence [4]. In our GDLD patients, the dysfunction of TACSTD2 is likely due to an in-frame deletion of 17 amino acids (L176_A192del) in TACSTD2. This in-frame deletion of TACSTD2 arises in a highly conserved region corresponding to the helix-turn-helix (HTH) configuration, and it is predicted to alter the ability of TACSTD2 to bind its target DNA sequences. The HTH is a short motif made up of an alpha-helix, a connecting turn, and a second helix, which specifically interacts with the DNA and is known as the recognition helix. The two alpha-helices extend from the domain surface and constitute a convex unit able to fit into the major groove of DNA [15][16][17]. Crystal structure analysis of the human TACSTD2 protein demonstrated that codons L176-A192 lie on the first alphahelix and connecting turn of the HTH motif. Therefore, it is likely that the in-frame deletion of these codons in the two brothers interferes with the correct folding of the HTH configuration in TACSTD2. GDLD could be due to the absence of functional TACSTD2 and/or the effects of the shortened protein.
Approximately 24 different GDLD-causing mutations in TACSTD2 have been documented to date. Most of the mutations reported have been missense and nonsense mutations. The mutations of nonsense and frame-shift are generally expected to have global detrimental effects on protein function. However, mutations causing amino acid alterations may be more informative with regard to the biochemical reason for development of a disease phenotype. With regards to GDLD, 8 of the 24 reported putative diseasecausing mutations produce amino acid alterations (excluding M1R, which affects initiation of protein synthesis). The novel 17 amino acid deletion mutation identified in our study was within a region between the TY and TM domains, signifying the importance of these regions in relation to TACSTD2 function. The function of the region between the TY and TM domains is unknown, but the mutations in the TY domain may affect its role as a protease inhibitor and thus affect amyloid deposition [18].
Nowadays, in vivo confocal microscopy and OCT have offered useful tools in the study of normal and diseased cornea. However, no data have been published with respect to the in vivo confocal microscopy and/or OCT appearance of this disease. Our morphological evaluation on GDLD by laser scanning in vivo confocal microscopy and Fourier-domain OCT further clarifies the pathological changes of this disease. In the presented case, which is clinically diagnosed as GDLD, laser scanning in vivo confocal microscopy and Fourierdomain OCT highlighted observations at the level of the epithelium and anterior stroma, was consistent with the results of the previous histological study of GDLD [19]. However, observations obtained by these two techniques are neither completely comparable with established clinical observations by the slit-lamp biomicroscope nor with the structural descriptions of established ex vivo microscopic techniques. In summary, our data add a novel mutation identified in two Chinese brothers with GDLD to the existing spectrum of TACSTD2 mutations.